Internal ribosome entry sites (IRESes) are cis-acting, structured RNA sequences that drive cap-independent translation initiation of a downstream template. This mechanism of starting protein synthesis is called internal initiation of translation. Viruses that use this mechanism include hepatitis C (HCV), hepatitis A (HAV), and foot and mouth disease (FMDV), among many others. In eukaryotic cells, this mechanism is used by some mRNAs associated with development, the cell cycle, growth and proliferation, apoptosis, and differentiation. The function of IRES RNAs depends on their tertiary structure, yet high-resolution structural data on these RNAs are limited. A detailed, complete structural understanding of a model IRES system is needed in order to understand the mechanism of internal initiation and possibly to target IRES RNAs with therapeutics. The cricket paralysis-like (CrPV) group of viral IRESes, like the HCV IRES, bind directly to the ribosome. Thus, they bypass the need for several protein initiation factors. Our preliminary results suggest that the CrPV-like IRESes adopt a compact three-dimensional fold, making them amenable to high-resolution structural and biophysical analysis. Hence, these IRESes represent an excellent model system for beginning to understand the structural basis of internal initiation. We will use analytical ultracentrifugation and small-angle x-ray scattering combined with mutagenesis and chemical and enzymatic probing to characterize the three-dimensional fold of the CRPV-like IRESes. We will use modification interference and nucleotide analog interference mapping to identify the determinants of the structure. We will correlate the IRES structure to the ability of the 40S subunit to bind using mutagenesis, modification and nucleotide analog interference mapping, and quantitative ribosome binding assays. Finally, we will solve the crystal structure of the complete IRES RNA, to include the domain critical for binding the 40S subunit as well as the domain proposed to occupy the P-site on the ribosome.